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The cell. 7. Cytosol. Cytoskeleton.

MICROTUBULES

Microtubules are a component of the cytoskeleton involved in the internal organization of eukaryote cells. They carry out many functions, such as contributing to the spatial organization of organelles, work as tracks for vesicular trafficking, are needed for cell division forming the mitotic spindle, help with cell movements, and are the skeleton of cilia and flagella.

1. Structure

Microtubules are long and relatively stiff tubules (Figures 1 and 3). Their wall is made up of many dimers of globular proteins: α- and β-tubulin (Figure 2), which are lined up in long rows known as protofilaments. Within a protofilament, there is no chemical bonds between adjoining tubulin dimers. A microtubule is usually composed of 13 protofilaments. α- and β-tubulin dimers are oriented in the same way, so that there is always α-tubulin in one end of the protofilament and β-tubulin in the other. It means that microtubules are polarized structures. The end formed by α-tubulin is known as minus end, and that formed by β-tubulin is known as plus end. New tubulin dimers are mainly added to the plus end, where the growth of the microtubule usually happens, although depolymerization also occurs. In the minus end, depolymerization prevails over polymerization. In this way, microtubules grow at plus end, while shrinkage occurs at the minus end. However, plus end is very dynamic, and alternation of polymerization anddepolymerization usually happens. In the minus end, depolymerization is more frequent.

 Microtubules
Figura 1. Organization of microtubules in an animal cell in culture.
 Protofilament
Figure 2. Organization of the tubulin dimers inside one protofilament. Note that α-tubulin is oriented to the minus end, whereas β-tubulin is toward the plus end.
Microtubules
Figure 3. Transmission electron microscopy image showing microtubules inside a dendrite of a neuron. Microtubules are oriented parallel to the long axis of the dendrite.

2. Dynamic instability

Microtubules are highly dynamic structures, continuously undergoing polymerization and depolymerization, mainly happening at the plus end. There is a steady exchage of tubulin dimers between the cytosol and microtubules. In a typical fibroblast, half of the available tubulin is free in the cytosol, and the other half are forming part of microtubules. The addition of new tubulin dimers to the plus end makes the microtubule to grow in length. The growth is stopped from time to time, and growth periods alternate with shrinkage periods. Depolymerization is sometimes so strong that the complete microtubule may disappear. However, it is more frequent a new polymerization (growing) period. The alternation between polymerizartion and depolymerization of microtubules is known as dynamic instability.

Free tubulin dimers are linked to two GTP molecules (Figure 4). After the joining of a tubulin dimer to the plus end of a microtubule, one GTP is hydrolyzed to ADP. If the rate of adding GTP-GTP-tubulin dimers to the plus end is faster than the hydrolysis rate, there will be always a group of tubulin dimers with GTP-GTP in the plus end, which is known as GTP-cap. GTP-cap stabilizes the plus end of the microtubule and boosts the polymerization. If the addition rate of new GTP-GTP-tubulin dimers is low, the hydrolysis rate may overcome the polymerization speed. This means that there are GTP-GDP-tubulin dimers at the plus end, which makes protofilaments to weakly adhere to one another. In this situation, a massive depolymerization starts. If the plus end is estabilized, the microbule grows again (Figure 4). GTP-ADP-tubulin dimers are released to the cytosol and quickly phosphorylated to GTP-GTP-tubulin dimers, which are ready to join to a microtubule plus end.

Dynamic instability
Figure 4. In this figure, GTP-GTP and GTP-GDP-tubulin dimers are depicted. In the cytosol, GTP-GDP-tubulin dimers are transformed in GTP-GTP-tubulin dimers, whereas GTP hydorlization occurs in the so-called hydrolysis zone of the microtubule wall. A microtubule shrinks when GTP-GDP-tubulin dimers are part of the plus end (there is no GTP cap), and it grows when GTP-GTP-tubulin dimers constitute the plus end (there is GTP cap).

3. MAPs

Microtubules do not directly interact much with other cell structures. However, there are microtubules associated proteins (MAPs) controlling the organization, stability, growing and other aspects of the microtubule behavior. MAPs may interact with the microtubule plus end, affecting the dynamic instability, either by boosting growth or depolymerization. Katanin develops a more drastic action because it breaks the microtubules down. MAPs also allow microtubules to interact with other cellular elements such as organelles or cytosolic molecules. Some substances that affect the polymerization or depolymerization of microtubules have been used as medicine drugs. For example, colchicine inhibit microtubule growth, whereas taxol strongly attach to microtubules preventing depolymerization.

4. Motor proteins

There are proteins that can get attached to microtubules and move either toward the plus end or the minus end. They are known as motor proteins. Dyneins and kinesins are the two motor protein families. Dyneins move toward the minus end of the microtubule, and kinesins toward the plus end. Both comprise a globular region that binds ATP and interacts with the microtubule, and a tail domain that recognizes and binds the cargo to be transported. ATP hydrolysis in globular domain changes the molecular conformation and makes the protein moves along the microtubule.

5. MTOCs

The concentration of cytosolic tubulin dimers is not high enough to spontaneously polymerizes and forms new microtubules. In the cell, there are molecular structures known as microtubule organizing centers (MTOCs), where microtubule are nucleated from. The minus end of the new microtubule is usually anchored to an MTOC, whereas the plus end grows the microtubule through the cytosol. Rings of γ-tubulin, located at the MOTCs, are molecular complexes that work as templates for the nucleation of new microtubules. Other proteins, such as TPX2 and XMAP125 also contribute to form new microtubules, either alone or cooperating with γ-tubulin rings.

The centrosome is the major MTOC in animal cells (Figure 5). It is the main responsible for the number, localization and spatial organization of microtubules. In most animal cells, during G1 and G0 phases of the cell cycle, there is one centrosome per cell found close to the nucleus. However, megakaryocytes contains multiple centrosomes, whereas muscle fibers lack centrosomes. Centrosome consists of two components: a couple of orthogonally oriented centrioles and a surrounding pericentriolar material. Each centriole is a cylindrical structure with a wall made up of 9 triplets of microtubules.

 Centrosome
Figure 5. In animal cells, centrosome is the main responsible for the microtubule scaffold nucleation and organization. Centrosome contains a couple of orthogonally arranged centrioles surrounded by the pericentriolar material. γ-tubulin rings, which are templates for microtubule nucleation, are located in the pericentriolar material.

There are many γ-tubulin molecules in the pericentriolar material arranged in rings, known as γ-tubulin rings, that nucleate microtubules. Centrioles, however, do not participate in the formation of microtubules nor in their spatial orientation, excepting the distal and subdistal appendages, which are structures attached to the mature centriole that can nucleate microtubules. The function of centrioles is still unknown. For example, plant cells lack centrioles, but they can segregate chromosomes, divide in two new cells, and organize their microtubules without any problem. Centrioles are similar to basal bodies, structures located in the basal part of cilia and flagella.

Centrosoma y ciclo celular
Cell cycle and centrosome

The centrosome is also important during the cell cycle because it contains many proteins involved in the progress of the cell cycle and in the organization of the mitotic spindle. For example, duplication of the centrosome before mitosis is essential to produce two "healthy" new cells.

There are other cellular places where microtubules can be nucleated. Blepharoplasts are molecular complexes found in plant cells and in some animal cells that can nucleate microtubules, and sometimes they can also form centrioles and centrosomes. Plant cells lack centrioles and do not form typical centrosomes, but they have γ-tubulin rings associated to the nuclear envelope, to blepharoplasts and scattered through the cytoplasm. Plant cells nucleate microtubules more frenquently in the peripheral cytoplasm. The main MTOC in yeasts is the polar body, which is inserted in the nuclear envelope (the mitotic spindle is intranuclear). There are other microtubule nucleators such as chromosomes, which can form a mitotic spindle without centrosomes. In plant cells, the mitotic spindle is made up of microtubles nucleated from the chromosomes. The cisterns of the Golgi apparatus nucleate microtubules that help to maintain the general organization of the organelle.

6. Function

Organization and movement of organelles. Microtubules are classified in two types: stable microtubules, located in cilia and flagella, and dynamic microtubules, located in the cytosol. Cytosolic microtubules, besides their role in the mitotic spindle formation and chromosome segregation, are also involved in the internal movement of organelles such as mitochondria, lysosomes, pigment inclusions, lipid drops, etcetera. They are also needed for vesicular trafficking. When cells in culture are observed at light microscopy, organelles show an alternation between quick movement and quiet periods. This behavior, known as saltatory movement, occurs when organelles are moving along the microtubules.

Microtubules are rather passive structures since they do not interact directly with organelles. The movement of organelles along the microtubules is produced by the activity of proteins known as motor proteins. There are two families of motor proteins: kinesins and dyneins. Both can "walk" along the external surface of microtubule walls. Kinesins go toward the plus end, whereas dyneins go toward the minus end. Their molecular structure has two globular domains and a tail domain. The globular domains bind ATP, generate the movement, and interact directly with the microtubule, whereas the tail selects and binds the cargoes (mainly organelles). ATP hydrolysis in the globular part leads to tridimensional molecular changes that allow the movement of the protein along the microtubule, dragging the cargo. Besides moving cargoes through the cytoplasm, motor proteins are also involved in the shape and localization of some organelles such as the Golgi complex and endoplasmic reticulum. Addition of colchicine depolymerizes the microtubular system of the cell, and both organelles collapse and divide in small vesicles scattered through the cytoplasm. When colchicine is removed and microtubule repolymerization occurs, both organelles get again the typical shape and cellular localization. These experiments indicate that there are proteins in the membranes of these organelles that are recognized by motor proteins.

Cilia and flagella are cellular structures protruding from the cell surface, contain microtubules, and are delimited by plasma membrane. Cells use cilia and flagella to move the surrounding liquid. They are also sensory structures. Cilia are shorter than flagella, are more numerous, and their movements propel the liquid parallel to the cell surface. Flagella move the surrounding liquid perpendicular to the cell surface.

Cilia and flagella are complex structures made up of more than 250 different proteins. Both have a central structure of microtubules known as axoneme, surrounded by the plasma membrane. Axoneme is formed by 9 outer pairs of microtubules, plus a central pair of microtubules. This organization is written down as 9x2+2. Microtubules of the axoneme grow from the microtubules of the basal body. Basal body have 9 triplets of microtubules (i.e. 9x3+0). The axoneme organization is strengthened by a scaffold of proteins. The outer pairs of microtubules of the axoneme are connected between each other by nexin proteins, whereas protein spokes connect the outer pairs with the central pair. The motor protein dynein is located between the outer pairs. Dynein is involved in the movement of cilia and flagella. The movement is a consequence of the sliding of one outer pair over another adjacent one, which forces the bending of the axoneme.

Some type of cilia are known as primary cilia. Primary cilia cannot move, are scarce, and sometimes appear alone in the cells. At least one cilium is present in most of the animal cells studied so far. Primary cilia bear in their membranes many receptors and ionic channels, so that a sensory role has been suggested. Nowadays, both primary and moving cilia are thought to develop a sensory function since both of them have receptors in their membranes.

Bibliography

Ohi R, Zanic M. 2016. Ahead of the curve: new insights into microtubule dynamics. F1000Research. 5:314.

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